The present invention contemplates compositions related to proteins which function in modulating animal physiology, e.g., controlling development, differentiation, trafficking, and physiology of mammalian cells, e.g., cells of a mammalian immune system. In particular, it provides purified genes, proteins, antibodies, and related reagents useful, e.g., to regulate activation, development, differentiation, and function of various cell types, particularly primate cells.
The circulating component of the mammalian circulatory system comprises various cell types, including red and white blood cells of the erythroid and myeloid cell lineages. See, e.g., Rapaport (1987) Introduction to Hematolocy (2d ed.) Lippincott, Philadelphia, Pa.; Jandl (1987) Blood: Textbook of Hematology, Little, Brown and Co., Boston, Mass.; and Paul (ed.) (1993) Fundamental Immunology (3d ed.) Raven Press, N.Y.
Growth factors, which are typically proteins, influence cellular proliferation and/or differentiation. Usually their effects are mediated through a cell membrane receptor. Some growth factors function via an autocrine signal. The growth factors have varying ranges of specificity of target cells. For example, certain factors play roles in oogenesis, see, e.g., Padgett, et al. (1987) Nature 325:81-84; Ferguson, et al. (1992) Cell 71:451-461; Staehling-Hampton, et al. (1994) Nature 372:783-786; and Panganiban, et al. (1990) Mol. Cell. Biol. 10:2669-2677, in embryogenesis, see, e.g., Xie, et al. (1994) Science 263:1756-1759; Raz, et al. (1993) Genes and Development 7:1937-1948; Brand, et al. (1994) Genes and Development 8:629-639; Goode, et al. (1992) Development 116:177-192; Livneh, et al. (1985) Cell 40:599-607; and Neuman-Silberberg, et al. (1993) Cell 75:164-174; and in morphogenesis, see, e.g., Heberlein, et al. (1993) Cell 75:913-926; Nellen, et al. (1994) Cell 78:225-237; Brummel, et al. (1994) Cell 78:251-261; and Penton, et al. (1994) Cell 78:239-250; of specific cell types or organs.
For some time, it has been known that the mammalian immune response is based on a series of complex cellular interactions, called the xe2x80x9cimmune network.xe2x80x9d Recent research has provided new insights into the inner workings of this network. While it remains clear that much of the response does, in fact, revolve around the network-like interactions of lymphocytes, macrophages, granulocytes, and other cells, immunologists now generally hold the opinion that soluble proteins, known as lymphokines, cytokines, or monokines, play a critical role in controlling these cellular interactions. Thus, there is considerable interest in the isolation, characterization, and mechanisms of action of cell modulatory factors, an understanding of which should lead to significant advancements in the diagnosis and therapy of numerous medical abnormalities, e.g., immune system and other disorders.
Lymphokines apparently mediate cellular activities in a variety of ways. They have been shown to support the proliferation, growth, and differentiation of the pluripotential hematopoietic stem cells into vast numbers of progenitors comprising diverse cellular lineages making up a complex immune system. These interactions between the cellular components are necessary for a healthy immune response. These different cellular lineages often respond in a different manner when lymphokines are administered in conjunction with other agents.
The chemokines are a large and diverse superfamily of proteins. The superfamily is subdivided into three branches, based upon whether the first two cysteines in the classical chemokine motif are adjacent (termed the xe2x80x9cCxe2x80x94Cxe2x80x9d branch) or spaced by an intervening residue (xe2x80x9cCxe2x80x94Xxe2x80x94Cxe2x80x9d), or a new branch which lacks two cysteines in the corresponding motif, represented by the chemokines known as lymphotactins. See, e.g., Schall and Bacon (1994) Current Opinion in Immunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allercy and Immunol. 109:97-109.
There is considerable interest in the isolation, characterization, and mechanisms of action of modulatory factors. Many factors have been identified which influence the differentiation process of precursor cells, or regulate the physiology or migration properties of specific cell types. These observations indicate that other factors exist whose functions in immune function were heretofore unrecognized. These factors provide for biological activities whose spectra of effects may be distinct from known differentiation or activation factors. The absence of knowledge about the structural, biological, and physiological properties of the regulatory factors which regulate cell physiology in vivo prevents the modification of the effects of such factors. Thus, medical conditions where regulation of the development or physiology of relevant cells is required remains unmanageable.
The present invention reveals the existence of a new family of Cysteine Rich Soluble Proteins (CRSPs). Their expression suggests a role in immunological function, particularly in inflammatory conditions. It is characterized in various rodent and human embodiments. Structural similarity to the defensins, along with expression levels, suggest a direct antimicrobial function, e.g., microbiostatic or microbiocidal.
The present invention comprises a composition of matter selected from: a substantially pure or recombinant C23 protein or peptide exhibiting identity over a length of at least 12 amino acids to SEQ ID NO: 2; a natural sequence C23 of SEQ ID NO: 2; or a fusion protein comprising C23 sequence. In certain embodiments, the protein comprises a segment exhibiting sequence identity to a corresponding portion of a C23, wherein: the identity is over at least about 15 amino acids; the identity is over at least 19 amino acids; or the identity is over at least 25 amino acids. In other-embodiments, the: C23 comprises a mature sequence of Table 1; or protein or peptide: is from a warm blooded animal selected from a mammal, including a primate; comprises at least one polypeptide segment of SEQ ID NO: 2; exhibits a plurality of portions exhibiting said identity; is a natural allelic variant of C23; has a length at least about 30 amino acids; exhibits at least two non-overlapping epitopes which are specific for a mammalian C23; exhibits a sequence identity at least about 90% over a length of at least about 20 amino acids to a primate C23; exhibits at least two non-overlapping epitopes which are specific for a primate C23; exhibits a sequence identity over a length of at least about 20 amino acids to a primate C23; is glycosylated; has a molecular weight of at least 7 kD with natural glycosylation; is a synthetic polypeptide; is attached to a solid substrate; is conjugated to another chemical moiety; is a 5-fold or less. substitution from natural sequence; or is a deletion or insertion variant from a natural sequence. Preferably, the composition can comprise: a sterile C23 protein or peptide; or the C2 protein or peptide and a carrier, wherein said carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.
Various fusion protein embodiments may comprise: mature protein sequence of Table 1; a detection or purification tag, including a FLAG, His6, or Ig sequence; or sequence of another cytokine or growth factor protein.
Certain kits will comprise a C23 protein or polypeptide, and: a compartment comprising said protein or polypeptide; and/or instructions for use or disposal of reagents in the kit.
Binding compounds are also provided, including a binding compound comprising an antigen binding portion from an antibody, which specifically binds to a natural C23 protein, wherein: the protein is a primate protein; the binding compound is an Fv, Fab, or Fab2 fragment; the binding compound is conjugated to another chemical moiety; or the antibody: is raised against a peptide sequence of a mature polypeptide of Table 1; is raised against a mature C23; is raised to a purified C23; is immunoselected; is a polyclonal antibody; binds to a denatured C23; exhibits a Kd to antigen of at least 30 xcexcM; is attached to a solid substrate, including a bead or plastic membrane; is in a sterile composition; or is detectably labeled, including a radioactive or fluorescent label. The invention further provides a kit comprising the binding compound, and: a compartment comprising said binding compound; and/or instructions for use or disposal of reagents in said kit. Preferably, the kit is capable of making a qualitative or quantitative analysis. Other embodiments include a composition comprising: a sterile binding compound of claim 7, or b) said binding compound and a carrier, wherein said carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.
Nucleic acid embodiments include an isolated or recombinant nucleic acid encoding a protein or peptide or fusion protein, wherein: the C family protein is from a mammal, including a primate; or the nucleic acid: encodes an antigenic peptide sequence of Table 1; encodes a plurality of antigenic peptide sequences of Table 1; exhibits at least about 80% identity to a natural cDNA encoding the segment; is an expression vector; further comprises an origin of replication; is from a natural source; comprises a detectable label; comprises synthetic nucleotide sequence; is less than 6 kb, preferably less than 3 kb; is from a mammal, including a primate; comprises a natural full length coding sequence; is a hybridization probe for a gene encoding said C family protein; or is a PCR primer, PCR product, or mutagenesis primer.
Certain cell embodiments which comprise a recombinant nucleic acid are provided, and preferably the cell is: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a mouse cell; a primate cell; or a human cell. Kits are provided, including those comprising such a nucleic acid, and: a compartment comprising the nucleic acid; a compartment further comprising a C23 protein or polypeptide; and/or instructions for use or disposal of reagents in the kit. Preferably, the kit is capable of making a qualitative or quantitative analysis.
Other nucleic acid embodiments include ones which: hybridize under wash conditions of 30xc2x0 C. and less than 2M salt to SEQ ID NO: 1; where the wash conditions are at 45xc2x0 C. and/or 500 mM salt; where the wash conditions are at 55xc2x0 C. and/or 150 mM salt; or exhibit at least about 85% identity over a stretch of at least about 30 nucleotides to a primate C23; or the identity is at least 90% and/or the stretch is at least 55 nucleotides; or where the identity is at least 95% and/or the stretch is at least 75 nucleotides.
The invention also provides a method of modulating physiology or development of a cell or tissue culture cells comprising contacting said cell with an agonist or antagonist of a C23.